Antineoplastic combinations

ABSTRACT

This invention provides the use of a combination of CCI-779 and EKB-569 in the treatment of neoplasms.

CROSS-REFERENCE TO OTHER REISSUE APPLICATION

More than one reissue application has been filed for the reissue of U.S.Pat. No. 6,617,333. This application is a divisional of reissueapplication Ser. No. 11/222,485, filed Sep. 8, 2005.

BACKGROUND OF THE INVENTION

This application claims priority from copending provisional applicationSerial No. 60/310,646, filed Aug. 7, 2001, the entire disclosure ofwhich is hereby incorporated by reference.

This invention relates to the use of combinations of rapamycin 42-esterwith 3-hydroxy-2-(hydroxymethyl)-2-methylpropionic acid (CCI-779) and4-dimethylamino-but-2-enoic acid[4-(3-chloro-4-fluoro-phenylamino)-3-cyano-7-ethoxy-quinolin-6-yl]-amide(EKB-569).

Rapamycin is a macrocyclic triene antibiotic produced by Streptomyceshygroscopicus, which was found to have antifungal activity, particularlyagainst Candida albicans, both in vitro and in vivo [C. Vezina et al.,J. Antibiot. 28, 721 (1975); S. N. Sebgal et al., J. Antibiot. 28, 727(1975); H. A. Baker et al., J. Antibiot. 31, 539 (1978); U.S. Pat. Nos.3,929,992; and 3,993,749]. Additionally, rapamycin alone (U.S. Pat. No.4,885,171) or in combination with picibanil (U.S. Pat. No. 4,401,653)has been shown to have antitumor activity.

The immunosuppressive effects of rapamycin have been disclosed in FASEB3, 3411 (1989). Cyclosporin A and FK-506, other macrocyclic molecules,also have been shown to be effective as immunosuppressive agents,therefore useful in preventing transplant rejection [FASEB 3, 3411(1989); FASEB 3, 5256 (1989); R. Y. Calne et al., Lancet 1183 (1978);and U.S. Pat. No. 5,100,899]. R. Martel et al. [Can. J. Physiol.Pharmacol. 55, 48 (1977)] disclosed that rapamycin is effective in theexperimental allergic encephalomyelitis model, a model for multiplesclerosis; in the adjuvant arthritis model, a model for rheumatoidarthritis; and effectively inhibited the formation of IgE-likeantibodies.

Rapamycin is also useful in preventing or treating systemic lupuserythematosus [U.S. Pat. No. 5,078,999], pulmonary inflammation [U.S.Pat. No. 5,080,899], insulin dependent diabetes mellitus [U.S. Pat. No.5,321,009], skin disorders, such as psoriasis [U.S. Pat. No. 5,286,730],bowel disorders [U.S. Pat. No. 5,286,731], smooth muscle cellproliferation and intimal thickening following vascular injury [U.S.Pat. Nos. 5,288,711 and 5,516,781], adult T-cell leukemia/lymphoma[European Patent Application 525,960 A1], ocular inflammation [U.S. Pat.No. 5,387,589], malignant carcinomas [U.S. Pat. No. 5,206,018], cardiacinflammatory disease [U.S. Pat. No. 5,496,832], and anemia [U.S. Pat.No. 5,561,138].

Rapamycin 42-ester with 3-hydroxy-2-(hydroxymethyl)-2-methylpropionicacid (CCI-779) is ester of rapamycin which has demonstrated significantinhibitory effects on tumor growth in both in vitro and in vivo models.The preparation and use of hydroxyesters of rapamycin, includingCCI-779, are disclosed in U.S. Pat. No. 5,362,718.

CCI-779 exhibits cytostatic, as opposed to cytotoxic properties, and maydelay the time to progression of tumors or time to tumor recurrence.CCI-779 is considered to have a mechanism of action that is similar tothat of sirolimus. CCI-779 binds to and forms a complex with thecytoplasmic protein FKBP, which inhibits an enzyme, mTOR (mammaliantarget of rapamycin, also known as FKBP12-rapamycin associated protein[FRAP]). Inhibition of mTOR's kinase activity inhibits a variety ofsignal transduction pathways, including cytokine-stimulated cellproliferation, translation of mRNAs for several key proteins thatregulate the G1 phase of the cell cycle, and IL-2-induced transcription,leading to inhibition of progression of the cell cycle from G1 to S. Themechanism of action of CCI-779 that results in the G1→S phase block isnovel for an anticancer drug.

In vitro, CCI-779 has been shown to inhibit the growth of a number ofhistologically diverse tumor cells. Central nervous system (CNS) cancer,leukemia (T-cell), breast cancer, prostate cancer, and melanoma lineswere among the most sensitive to CCI-779. The compound arrested cells inthe G1 phase of the cell cycle.

In vivo studies in nude mice have demonstrated that CCI-779 has activityagainst human tumor xenographs of diverse histological types. Gliomaswere particularly sensitive to CCI-779 and the compound was active in anorthotopic glioma model in nude mice. Growth factor(platelet-derived)-induced stimulation of a human glioblastoma cell linein vitro was markedly suppressed by CCI-779. The growth of several humanpancreatic tumors in nude mice as well as one of two bread cancer linesstudied in vivo also was inhibited by CCI-779.

Protein tyrosine kinases are a class of enzymes that catalyze thetransfer of a phosphate group from ATP or GTP to tyrosine residuelocated on protein substrates. Protein tyrosine kinases clearly play arole in normal cell growth. Many of the growth factor receptor proteinsfunction as tyrosine kinases and it is by this process that they effectsignaling. The interaction of growth factors with these receptors is anecessary event in normal regulation of cell growth. However, undercertain conditions, as a result of either mutation or overexpression,these receptors can become deregulated; the result of which isuncontrolled cell proliferation which can lead to tumor growth andultimately to the disease known as cancer [Wilks A. F., Adv. CancerRes., 60, 43 (1993) and Parsons, J. T.; Parsons, S. J., ImportantAdvances in Oncology, DeVita V. T. Ed., J. B. Lippincott Co., Phila., 3(1993)]. Among the growth factor receptor kinases and theirproto-oncogenes that have been identified and which are targets of thecompounds of this invention are the epidermal growth factor receptorkinase (EGF-R kinase, the protein product of the erbB oncogene), and theproduct produced by the erbB-2 (also referred to as the neu or HER2)oncogene. Since the phosphorylation event is a necessary signal for celldivision to occur and since overexpressed or mutated kinases have beenassociated with cancer, an inhibitor of this event, a protein tyrosinekinase inhibitor, will have therapeutic value for the treatment ofcancer and other diseases characterized by uncontrolled or abnormal cellgrowth. For example, overexpression of the receptor kinase product ofthe erbB-2 oncogene has been associated with human breast and ovariancancers [Slamon, D. J., et. al., Science, 244, 707 (1989) and Science,235, 1146 (1987)]. Deregulation of EGF-R kinase has been associated withepidermoid tumors [Reiss, M., et. al., Cancer Res., 51, 6254 (1991)],breast tumors [Macias, A., et. al., Anticancer Res., 7, 459 (1987)], andtumors involving other major organs [Gullick, W. J., Brit. Med. Bull.,47, 87 (1991)]. Because of the importance of the role played byderegulated receptor kinases in the pathogenesis of cancer, many recentstudies have dealt with the development of specific PTK inhibitors aspotential anti-cancer therapeutic agents [some recent reviews: Burke, T.R., Drugs Future, 17, 119 (1992) and Chang, C. J.; Geahlen, R. L., J.Nat. Prod., 55, 1529 (1992)].

4-Dimethylamino-but-2-enoic acid[4-(3-chloro-4-fluoro-phenylamino)-3-cyano-7-ethoxy-quinolin-6-yl]-amide(EKB-569) is an EGFR kinase inhibitor which has significant inhibitoryeffects on tumor growth in both in vitro and in vivo models. Thepreparation and use of EGFR kinase inhibitors, such as EKB-569, aredisclosed in U.S. Pat. No. 6,002,008.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows cytotoxicity curves of EKB-569, CCI-779, and combinationsof EKB-569+CCI-779 in HCT116 cells.

FIG. 2 shows isobolograms (at the 50% effect level) of a EKB-569+CCI-779combination.

FIG. 3 shows isobolograms for EKB-569+CCI-779 combinations derived fromdifferent endpoints ranging from 50-65%.

FIG. 4 shows a 3-dimensional analysis of the synergistic interaction ofa EKB-569+CCI-779 combination.

FIG. 5 shows a contour plot of the 3-dimensional synergy plot of aEKB-569+CCI-779 combination.

DESCRIPTION OF THE INVENTION

This invention provides the use of combinations of CCI-779 and EKB-569as antineoplastic combination chemotherapy. In particular, thesecombinations are useful in the treatment of renal cancer, soft tissuecancer, breast cancer, neuroendocrine tumor of the lung, cervicalcancer, uterine cancer, head and neck cancer, glioma, non-small lungcell cancer, prostate cancer, pancreatic cancer, lymphoma, melanoma,small cell lung cancer, ovarian cancer, colon cancer, esophageal cancer,gastric cancer, leukemia, colorectal cancer, and unknown primary cancer.This invention also provides combinations of CCI-779 and EKB-569 for useas antineoplastic combination chemotherapy, in which the dosage ofeither CCI-779 or EKB-569 or both are used in subtherapeuticallyeffective dosages.

As used in accordance with this invention, the term “treatment” meanstreating a mammal having a neoplastic disease by providing said mammalan effective amount of a combination of CCI-779 and EKB-569 with thepurpose of inhibiting growth of the neoplasm in such mammal, eradicationof the neoplasm, or palliation of the mammal.

As used in accordance with this invention, the term “providing,” withrespect to providing the combination, means either directlyadministering the combination, or administering a prodrug, derivative,or analog of one or both of the components of the combination which willform an effective amount of the combination within the body.

The preparation of CCI-779 is described in U.S. Pat. No. 5,362,718,which is hereby incorporated by reference. An improved preparation ofCCI-779 is disclosed in U.S. patent application Ser. No. 09/670,358, nowU.S. Pat. No. 6,277,983, which is hereby incorporated by reference. WhenCCI-779 is used as an antineoplastic agent, it is projected that initiali.v. infusion dosages will be between about 0.1 and 100 mg/m² whenadministered on a daily dosage regimen (daily for 5 days, every 2-3weeks), and between about 0.1 and 1000 mg/m² when administered on a onceweekly dosage regimen. Oral or intravenous infusion are the preferredroutes of administration, with intravenous being more preferred.

EKB-569 can be prepared according to the procedures described in U.S.Pat. No. 6,002,008, which is incorporated by reference. Preferredprocedures for the preparation of EKB-569 are provided herein. WhenEKB-569 is used as an antineoplastic agent it is projected that theinitial oral dosage will be between 1 and 100 mg per day. Depending onpatient tolerance, EKB-569 can be administered daily for a treatmentperiod, such as 14 days, followed by a rest period (no drugadministered), or can be administered on a continuous basis for a longertreatment period (for example, 6 months or longer).

The antineoplastic activity of the CCI-779 plus EKB-569 combination wasconfirmed in in vitro standard pharmacological test procedure; thefollowing briefly describes the procedure used and the results obtained.

Cell Proliferation Procedure—HCT 116 colon adenocarcinoma cells weremaintained in RPMI 1640 medium (Life Technologies, Inc., Gaithersburg,Md.) supplemented with 10% fetal bovine serum (FBS, Life Technologies)and 50 μg/ml gentamicin (Life Technologies) under 7% CO₂ at 37° C. Cellswere plated in 96-well microtiter dishes (6000 cells/well) in 200 μlRPMI 1640 medium containing 5% FBS and 50 μg/ml gentamicin and incubatedovernight at 37° C. Compound dilutions were prepared in the same medium,at 5× final concentration, and 50 μl of the drug dilution was added tothe cell-containing wells. For studies involving combinations of twodrugs, serial dilutions of one compound were prepared in the presence ofa fixed dose of a second compound. Alternatively, a checkerboarddilution series was employed. Cells were cultured for three days in thepresence of the drugs. Untreated cells were included as controls. Thepercentage of surviving cells was determined using sulforhodamine B(SRB, Sigma-Aldrich, St Louis, Mo.), a protein binding dye. Cellularprotein was precipitated in each well by the addition of 50 μl of 50%cold trichloroacetic acid. After 1 hour, the plates were washedextensively in water and dried. SRB dye reagent (0.4% SRB in 1% aceticacid, 80 μl per well) was added and plates were kept at room temperaturefor ten minutes. Plates were then washed thoroughly in 1% acetic acidand dried. Cell-associated dye was dissolved in 10 mM Tris (150 μl) andthe absorbance was read at 540 nm in a microtiter plate reader. Theconcentration of compound that caused a fixed percentage inhibition ofgrowth was determined by plotting cell survival (relative to untreatedcells) against the compound dose.

Synergy Evaluation—Isobolograms were used to study the interaction oftwo pharmacological agents. Here, the concentration of each drug alonewhich produces a certain endpoint (e.g 50% inhibition of cell growth,IC₅₀), is plotted on the two graphical axes. The straight lineconnecting the two points represents equally effective concentrations ofall combinations of the two drugs if the interaction is purely additive.A shift of the isobologram to the left of the predicted cytoxicity(curve with concave side up) represents a synergistic interaction.Conversely, a shift to the right (isobologram with the convex side up)represents an antagonistic interaction. When isobolograms for differentendpoints were plotted on the same graph, the concentration of each drugwas expressed as the fraction of the concentration of each drug alonethat produced the same effect. This produces a symmetrical isobologramwith unit-less measures on each axis, and allows a direct comparison ofdifferent endpoints.

A second model for studying drug interactions was proposed by Prichardand Shipman [Antiviral Research. 14:181-206 (1990)]. This is a3-dimensional model: one for each drug and the third for the biologicaleffect. Theoretical additive interactions are calculated from theindividual dose-response curves, based on a dissimilar sites model ofadditivity (Bliss independence). The calculated additive surface,representing predicted cytotoxicity is subtracted from the experimentalsurface to reveal areas of enhanced toxicity (synergy) or reducedtoxicity (antagonism). The resulting surface appears as a horizontalplane at 0% inhibition above the calculated additive surface, if theinteraction is additive. Peaks and valleys deviating from this plane areindicative of synergy and antagonism, respectively. MacSynergyll, aMicrosoft Excel-based software was used to perform all calculationsautomatically. This spreadsheet calculates the theoretical additiveinteractions, and locates and quantifies synergistic or antagonisticinteraction that are significant at the 95% confidence levels. Theresults were plotted as a 3-dimensional plot, or as a contour plot.

Results—HCT 116 cells were chosen as they express low, but detectablelevels of EGFR, and are sensitive to inhibition by EGFR inhibitors. Thecells are somewhat resistant to CCI-779, but are inhibited by high doses(5-10 μg/ml) of this drug. HCT-116 cells were cultured in the presenceof EKB-569 alone, CCI-779 alone, or a dilution series of EKB-569 withfixed doses of CCI-779. Following growth for 3 days, cell survival wasdetermined using the SRB test procedure. Cytotoxicity curves are shownin FIG. 1. EKB-569 produced an IC₅₀ value of 0.31 μg/ml in HCT116 cells.When this compound was combined with 2.08 μg/ml CCI-779 (which caused41% inhibition of growth when administered alone), the IC₅₀ value isreduced to 0.03 μg/ml, a 10-fold decrease. When combined with 0.026μg/ml CCI-779 (which alone inhibits cell proliferation by 36%), the IC₅₀value dropped to 0.051 μg/ml, a 6-fold decrease. Similar results wereobserved when dose-response curves were produced with CCI-779 in thepresence of fixed doses of EKB-569. To identify the nature of this druginteraction, isobolograms (at 50% effect level) of the combination ofEKB-569 and CCI-779 were generated (FIG. 2). The isobologram was deeplyindented with the concave side up, indicating a substantial synergisticinteraction between the two drugs. At the most synergistic point, 0.03μg/ml of EKB-569 combined with 0.077 μg/ml CCI-779 was iso-effectivewith 0.31 μg/ml of EKB-569 alone or 4.3 μg/ml CCI-779 alone (IC₅₀ foreach drug alone). Thus, a 10-fold reduction in the dose of EKB-569 and a50-fold reduction in the dose of CCI-779 was required to inhibit cellproliferation by 50% when the drugs were combined, compared to eitherdrug alone. Isobolograms derived from different endpoints, ranging from50 to 65% were also examined. As shown in FIG. 3., the isobologramsproduced were almost superimposable, indicating synergy at all effectlevels tested.

The interaction between EKB-569 and CCI-779 was also evaluated using a3-dimensional analysis. Here, pharmacological interactions are presentedin a 3-dimensional plot with the plane at 0% representing additiveinteraction, and peaks and valleys representing areas of synergy orantagonism, respectively, between the two drugs. In FIG. 4, thecombination of EKB-569 and CCI-779 resulted in a broad area ofsynergistic interaction, consistent with the results shown in theisobologram studies. A contour plot of the 3-dimensional synergy plotfacilitates the identification of the concentrations of drugs at whichgreatest synergistic toxicity occurs (FIG. 5). A broad area of synergywas observed at 0.0005 to 3 μg/ml CCI-779 and 0.16 to 0.4 μg/ml EKB-569.Within this area, two peaks of maximum synergy occurred at 0.0005 to0.003 μg/ml and 0.05 to 0.3 μg/ml of CCI-779 and 0.25 to 0.37 μg/mlEKB-569.

Based on the results of these standard pharmacological test procedures,combinations of CCI-779 plus EKB-569 acted synergistically together, andare useful as antineoplastic therapy. More particularly, thesecombinations are useful in the treatment of renal carcinoma, soft tissuesarcoma, breast cancer, neuroendocrine tumor of the lung, cervicalcancer, uterine cancer, head and neck cancer, glioma, non-small celllung cancer, prostate cancer, pancreatic cancer, lymphoma, melanoma,small cell lung cancer, ovarian cancer, colon cancer, esophageal cancer,gastric cancer, leukemia, colorectal cancer, and unknown primary cancer.As these combinations contain at least two active antineoplastic agents,the use of such combinations also provides for the use of combinationsof each of the agents in which one or both of the agents is used atsubtherapeutically effective dosages, thereby lessening toxicityassociated with the individual chemotherapeutic agent.

In providing chemotherapy, multiple agents having different modalitiesof action are typically used as part of a chemotherapy “cocktail.” It isanticipated that the combinations of this invention will be used as partof a chemotherapy cocktail that may contain one or more additionalantineoplastic agents depending on the nature of the neoplasia to betreated. For example, this invention also covers the use of theCCI-779/EKB-923 combination used in conjunction with otherchemotherapeutic agents, such as antimetabolites (i.e., 5-fluorouracil,floxuradine, thioguanine, cytarabine, fludarabine, 6-mercaptopurine,methotrexate, gemcitabine, capecitabine, pentostatin, trimetrexate, orcladribine); DNA crosslinking and alkylating agents (i.e., cisplatin,carboplatin, streptazoin, melphalan, chlorambucil, carmustine,methclorethamine, lomustine, bisulfan, thiotepa, ifofamide, orcyclophosphamide); hormonal agents (i.e., tamoxifen, roloxifen,toremifene, anastrozole, or letrozole); antibiotics (i.e., plicamycin,bleomycin, mitoxantrone, idarubicin, dactinomycin, mitomycin,doxorubicin or daunorubicin); immunomodulators (i.e., interferons, IL-2,or BCG); antimitotic agents (i.e., estramustine, paclitaxel, docetaxel,vinblastine, vincristine, or vinorelbine); topoisomerase inhibitors(i.e., topotecan, irinotecan, etoposide, or teniposide.); and otheragents (i.e., hydroxyurea, trastuzumab, altretamine, retuximab,L-asparaginase, or gemtuzumab ozogamicin).

As used in this invention, the combination regimen can be givensimultaneously or can be given in a staggered regimen, with CCI-779being given at a different time during the course of chemotherapy thanEKB-923. This time differential may range from several minutes, hours,days, weeks, or longer between administration of the two agents.Therefore, the term combination does not necessarily mean administeredat the same time or as a unitary dose, but that each of the componentsare administered during a desired treatment period. The agents may alsobe administered by different routes. For example, in the combination ofCCI-779 plus EKB-569, it is anticipated that the CCI-779 will beadministered orally or parenterally, with parenterally being preferred,while the EKB-569 may be administered parenterally, orally, or by otheracceptable means. These combination can be administered daily, weekly,or even once monthly. As typical for chemotherapeutic regimens, a courseof chemotherapy may be repeated several weeks later, and may follow thesame timeframe for administration of the two agents, or may be modifiedbased on patient response.

As typical with chemotherapy, dosage regimens are closely monitored bythe treating physician, based on numerous factors including the severityof the disease, response to the disease, any treatment relatedtoxicities, age, health of the patient, and other concomitant disordersor treatments.

Based on the results obtained with the CCI-779 plus EKB-569combinations, it is projected that the initial i.v. infusion dosage ofCCI-779 will be between about 0.1 and 100 mg/m², with between about 2.5and 70 mg/m² being preferred. It is also preferred that the CCI-779 beadministered by i.v., typically over a 30 minute period, andadministered about once per week. The initial daily dosages of EKB-569will be between about 1 and 100 mg, with between 5 and 75 mg beingpreferred. After one or more treatment cycles, the dosages can beadjusted upwards or downwards depending on the results obtained and theside effects observed.

Oral formulations containing the active compounds of this invention maycomprise any conventionally used oral forms, including tablets,capsules, buccal forms, troches, lozenges and oral liquids, suspensionsor solutions. Capsules may contain mixtures of the active compound(s)with inert fillers and/or diluents such as the pharmaceuticallyacceptable starches (e.g. corn, potato or tapioca starch), sugars,artificial sweetening agents, powdered celluloses, such as crystallineand microcrystalline celluloses, flours, gelatins, gums, etc. Usefultablet formulations may be made by conventional compression, wetgranulation or dry granulation methods and utilize pharmaceuticallyacceptable diluents, binding agents, lubricants, disintegrants, surfacemodifying agents (including surfactants), suspending or stabilizingagents, including, but not limited to, magnesium stearate, stearic acid,talc, sodium lauryl sulfate, microcrystalline cellulose,carboxymethylcellulose calcium, polyvinylpyrrolidone, gelatin, alginicacid, acacia gum, xanthan gum, sodium citrate, complex silicates,calcium carbonate, glycine, dextrin, sucrose, sorbitol, dicalciumphosphate, calcium sulfate, lactose, kaolin, mannitol, sodium chloride,talc, dry starches and powdered sugar. Preferred surface modifyingagents include nonionic and anionic surface modifying agents.Representative examples of surface modifying agents include, but are notlimited to, poloxamer 188, benzalkonium chloride, calcium stearate,cetostearyl alcohol, cetomacrogol emulsifying wax, sorbitan esters,colloidal silicon dioxide, phosphates, sodium dodecylsulfate, magnesiumaluminum silicate, and triethanolamine. Oral formulations herein mayutilize standard delay or time release formulations to alter theabsorption of the active compound(s). The oral formulation may alsoconsist of administering the active ingredient in water or a fruitjuice, containing appropriate solubilizers or emulsifiers as needed.

In some cases it may be desirable to administer the compounds directlyto the airways in the form of an aerosol.

The compounds may also be administered parenterally orintraperitoneally. Solutions or suspensions of these active compounds asa free base or pharmacologically acceptable salt can be prepared inwater suitably mixed with a surfactant such as hydroxy-propylcellulose.Dispersions can also be prepared in glycerol, liquid polyethyleneglycols and mixtures thereof in oils. Under ordinary conditions ofstorage and use, these preparation contain a preservative to prevent thegrowth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases, the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (e.g., glycerol, propylene glycol and liquidpolyethylene glycol), suitable mixtures thereof, and vegetable oils.

For the purposes of this disclosure, transdermal administrations areunderstood to include all administrations across the surface of the bodyand the inner linings of bodily passages including epithelial andmucosal tissues. Such administrations may be carried out using thepresent compounds, or pharmaceutically acceptable salts thereof, inlotions, creams, foams, patches, suspensions, solutions, andsuppositories (rectal and vaginal).

Transdermal administration may be accomplished through the use of atransdermal path containing the active compound and a carrier that isinert to the active compound, is non toxic to the skin, and allowsdelivery of the agent for systemic absorption into the blood stream viathe skin. The carrier may take any number of forms such as creams andointments, pastes, gels, and occlusive devices. The creams and ointmentsmay be viscous liquid or semisolid emulsions of either the oil-in-wateror water-in-oil type. Pastes comprised of absorptive powders dispersedin petroleum or hydrophilic petroleum containing the active ingredientmay also be suitable. A variety of occlusive devices may be used torelease the active ingredient into the blood stream such as asemi-permeable membrane covering a reservoir containing the activeingredient with or without a carrier, or a matrix containing the activeingredient. Other occlusive devices are known in the literature.

Suppository formulations may be made from traditional materials,including cocoa butter, with or without the addition of waxes to alterthe suppository's melting point, and glycerin. Water soluble suppositorybases, such as polyethylene glycols of various molecular weights, mayalso be used.

The following provides the preparation of EKB-569 from commerciallyavailable starting materials or starting materials that can be madeaccording to available literature procedures.

Preparation of 4-dimethylaminocrotonic acid from TMS-4-bromocrotonate

211 ml dimethylamine (2M in THF, 0.422 moles) was added drop-wise to asolution of 50 g TMS-4-bromocrotonate (0.211 moles, 75.9% by GC-MS) in250 ml of THF at 0-50 C. under N₂. The reaction mixture was stirred atroom temperature for 30 minutes. A white solid by-product was filteredoff. 2 ml water was added to the filtrate followed by seeding. Thecrystals formed were filtered and washed with ether to give 18.3 g (fromtwo crops) off-white solid product. Yield was 67.2% (98% purity byGC-MS, NMR was consistent with the structure).

Preparation of methyl 4-dimethylaminocrotonate frommethyl-4-bromocrotonate

120 ml dimethylamine (2M in THF, 0.24 moles) was added drop-wise to asolution of 20 g methyl 4-bromocrotonate (85% purity, 0.095 moles) in150 ml of THF at 0-50 C. under N₂. The reaction mixture was stirred for15 minutes at room temperature. TLC (9:1 CH₂Cl₂:MeOH with few drops ofEt₃N) showed residual methyl 4-bromocrotonate. The reaction mixture washeated to 40-450 C. for 15 minutes. A white solid by-product wasfiltered off. The filtrate was evaporated to give a yellow oil (14 g).The yellow oil was dissolved in 100 ml CH₂Cl₂ and washed with H₂O twice.The aqueous layer was back extracted with 100 ml CH₂Cl₂. The CH₂Cl₂layers were combined, dried over MgSO₄ and filtered. The filtrate wasevaporated to give an oil (12 g). Yield was 88%. NMR indicated desiredproduct with trace methyl 4- bromocrotonate.

Preparation of Methyl 4-N,N-dimethlaminocrotonate hydrochloride on largescale

A 3 L flask was charged with tetrahydrofuran (0.71 kg, 0.80 L). Methyl4-bromocrotonate (0.20 kg, 0.13 L, d=1.522 g/mL) was added and rinsedwith tetrahydrofuran (0.18 kg, 0.20 L). The solution was stirred andcooled to 0-10° C. An additional funnel was charged with a solution ofdimethylamine in tetrahydrofuran and added over (1 h 15 min) keeping thetemperature at 0-10° C. The mixture was stirred for a minimum of 30 minsand checked for reaction completion by TLC. The reaction was completewhen there is ≦2% detectable starting material (methyl 4-bromocrotonate)present. The mixture was filtered cold on a Buchner funnel into a 3 Lmulti-neck flask, rinsed with pre-chilled (0-10° C.) tetrahydrofuran(2×0.18 kg, 2×0.20 L), and suction maintained until dripping stops. Theflask was equipped with an agitator, thermometer, and a setup for vacuumdistillation. The solution was concentrated by distillation under areduced pressure of (125-200 mm Hg) and at a maximum pot temperature of(40° C.) to a pot volume of (200 mL). Isopropanol (0.22 kg, 0.28 L) wasadded and the mixture cooled to 0-10° C. The distillation stillhead wasreplaced with an addition funnel charged with a solution of HCl inisopropanol, which was added over 45 min until pH of 2.0-3.0 wasreached, while maintaining a temperature 0-10° C. The mixture was heldfor a minimum 30 min, and fileted cold on a Buchner funnel, rinsed withisopropanol (2×0.12 kg, 2×0.15 L). The filter cake was dammed andsuction maintained until dripping stopped. The product was dried in avacuum oven at 50° C. and 10 mm Hg for 18-20 h.

Preparation of 4-dimethylaminocrotonic acid hydrochloride from methyl4-dimethylaminocrotonate

A NaOH solution (3.35 g in 25 ml H₂O, 0.084 moles) was added drop-wiseto a solution of 12 g methyl 4-dimethylaminocrotonate (0.084 moles) in100 ml MeOH at room temperature. The reaction mixture was heated to40-45° C. for 1 hour then cooled to room temperature. The pH wasadjusted to 1-2 with 5 N HCl. The mixture was concentrated to a thickoil which was triturated with dehydrated alcohol to form a solid. Thesolid by-product was filtered off. The filtrate was evaporated to an oilwhich was triturated with IPA. Seven (7.0) g of white solid product wasobtained. Yield was 50% with the purity 86.3% by GC-MS.

Preparation of 4-N,N-dimethylaminocrotonic acid hydrochloride on largescale

A 2 L multi-neck flask was equipped with agitator, thermometer, additionfunnel, and nitrogen protection. The flask was charged with ethanol(0.39 kg, 0.50 L). Methyl 4-N,N-dimethylamino crotonate hydrochloride(0.125 kg) was added and rinsed with ethanol (0.10 kg, 0.125 L). Thesuspension was stirred and cooled to 0-10° C. The addition funnel wascharged with sodium hydroxide (50%) (0.11 kg, 0.072 L, d=1.53 g/mL) andaddd over 20 min keeping the temperature at 0-10° C. A slight exothermwas observed and the mixture turned yellow. The mixture was stirred fora minimum of 15 min, and then warmed to 18-22° C., and held for aminimum of 4 h. The reaction was checked for completion by TLC. Thereaction is complete when there is ≦2% detectable starting material(methyl 4-N,N-dimethylaminocrotonate hydrochloride) present. The mixturewas cooled to 0-10° C. An addition funnel was charged with a solution ofHCl in isopropanol and added over 40 min until pH 2.0-3.0 was attained,while maintaining the pot temperature of 0-10° C. The mixture wassturred for a minimum of 30 min, and filtered cold on a Buchner funnelinto a 2 L multi-neck flask, rinsed with cold ethanol (0-10° C.) (2×0.05kg, 2×0.063 L) with suction maintained until dripping stops. The flaskwas equpped with an agitator, thermometer, and setup for vacuumdistillation. Solvent was removed under a reduced pressure of 50-100 mmHg and at a maximum pot temperature of (40° C.) to a pot volume of160-180 mL. Isopropanol (0.049 kg, 0.063 L) was added, and the mixturewarmed to 35-40° C. over 10 min. Acetone (0.10 kg, 0.13 L) was addedover 20 min while maintaining the pot temperature at 35-40° C. Themixture was seeded and cooled to ambient temperature 20-25° C., and heldthere for a minimum of 12-18 h. The mixture was cooled to 0-10° C., heldthere for a minimum of 1 h. A mixture of isopropanol (0.049 kg, 0.063 L)and acetone (0.10 kg, 0.13 L) was prepared, stirred to homogenize, andcooled to 0-10° C. The mixture was filtered cold on a Buchner funnel,rinsed with isopropanol/acetone (2×0.074 kg, 2×0.096 L), and the filtercaked dammed while maintaining suction until dripping stopped. Theproduct was dried in a vacuum oven at 50° C. and 10 mm Hg for 18-20 h.

Preparation of 4-dimethylaminocrotonyl anilide from4-dimethylaminocrotonic acid hydrochloride

Thionyl chloride (0.36 ml, 0.005 moles) was added dropwise to a solutionof 0.33 g 4-dimethylaminocrotonic acid hydrochloride (0.002 moles) in 15ml CH₂Cl₂ containing 2 drops of DMF at 0° C. under N2. The reactionmixture was refluxed for 30 min. Then 0.72 ml aniline (0.008 moles) wasadded drop-wise to the reaction mixture at 0° C. and stirred for 1 hourat room temperature. A solid by-product was filtered. The filtrate wasevaporated to give an oil (0.6 g). GC-MS data shows that the oil is11.7% 4-dimethylaminocrotonic acid hydrochloride and 85% of desiredproduct.

Preparation and isolation of 4-N,N-dimethylaminocrotonoylchloridehydrochloride

A well stirred suspension 4-dimethylaminocrotonic acid hydrochloride(5.0 g, 30 mmol) in cold (0° C.) THF (40 mL) and DMF (2 pipet drops) wastreated with oxalyl chloride (3.15 mL, 36 mmol). The mixture was stirredat 20-25° C. for 3 h then cooled to 0° C. and held for 30 min. Thesolids were collected on Buchner funnel (under a blanket of nitrogen)and washed with cold (0° C.) THF (3×5 mL). The product was dried undervacuum (−1 torr) at 40-50 2° C. for 3 h to give 4.0 g of4-dimethylaminocrotonoyl chloride hydrochloride. This material ischaracterized as its methyl ester by treatment of the solid withmethanol.

Alternatively, the title compound can be prepared in CH₃CN and useddirectly for the coupling step:

Preparation of EKB-569

A 3 L multi-neck flask was equipped with an agitator, thermometer, diptube, and nitrogen protection. The flask was charged with N-methylpyrrolidinone (0.77 kg, 0.75 L, d=1.033 g/mL). At ambient temperature,4-[3-chloro-4-fluorophenyl]amino-6-amino-3-cyano-7-ethoxy quinoline(0.0748 kg) [see, U.S. Pat. No. 6,002,008] was added and the mixturestirred while heating to 40-45° C. and hold for 15 min. The flask wascooled to 0-10° C. The mixture containing 4-N,N-dimethylaminocrotonoylchloride hydrochloride was transferred via dip tube and positivenitrogen pressure to the 3 L flask over 30-45 min, while maintaining0-10° C. The mixture was kept at 0-10° C. for a minimum of 2 h. Thereaction was checked for completion by HPLC. The reaction is completewhen there is ≦2% of the starting material(4-[3-chloro-4-fluorophenyl]amino-6-amino-3-cyano-7-ethoxy quinoline)present. A 12 L multi-neck flask equipped with agitator, thermometer,dip tube, and nitrogen protection was charged with water (2.61 kg, 2.61L). Sodium bicarbonate (0.209 kg) was added and stirred until a solutionwas obtained. The solution was cooled to 20-24° C. The NMP-CH₃CN mixturewas transferred, via dip tube and positive nitrogen pressure, to the 12L flask over 45-60 min, while maintaining 20-24° C. The mixture wasmaintained at 20-24° C. for a minimum of 1 h, and filtered on a Buchnerfunnel, and rinsed with water (3×0.40 kg, 3×0.40 L) with suction beingmaintained until dripping stops. The product was dried in a vacuum ovenat 50° C. and 10 mm Hg for 28-30 h to give 78.5 g (86% yield) ofproduct.

1. A method of treating a neoplasm in a mammal in need thereof, whichcomprises providing to said mammal synergistically effective amount of acombination comprising CCI-779 and EKB-569.
 2. The method according toclaim 1, wherein the neoplasm is renal cancer.
 3. The method accordingto claim 1, wherein the neoplasm is soft tissue sarcoma.
 4. The methodaccording to claim 1, wherein the neoplasm is breast cancer.
 5. Themethod according to claim 1, wherein the neoplasm is a neuroendocrinetumor of the lung.
 6. The method according to claim 1, wherein theneoplasm is cervical cancer.
 7. The method according to claim 1, whereinthe neoplasm is uterine cancer.
 8. The method according to claim 1,wherein the neoplasm is a head and neck cancer.
 9. The method accordingto claim 1, wherein the neoplasm is glioma.
 10. The method according toclaim 1, wherein the neoplasm is non-small cell lung cancer.
 11. Themethod according to claim 1, wherein the neoplasm is prostate cancer.12. The method according to claim 1, wherein the neoplasm is pancreaticcancer.
 13. The method according to claim 1, wherein the neoplasm islymphoma.
 14. The method according to claim 1, wherein the neoplasm ismelanoma.
 15. The method according to claim 1, wherein the neoplasm issmall cell lung cancer.
 16. The method according to claim 1, wherein theneoplasm is ovarian cancer.
 17. The method according to claim 1, whereinthe neoplasm is colon cancer.
 18. The method according to claim 1,wherein the neoplasm is esophageal cancer.
 19. The method according toclaim 1, wherein the neoplasm is gastric cancer.
 20. The methodaccording to claim 1, wherein the neoplasm is leukemia.
 21. The methodaccording to claim 1, wherein the neoplasm is colorectal cancer.
 22. Themethod according to claim 1, wherein the neoplasm is unknown primarycancer.
 23. A method of treating a neoplasm in a mammal in need thereof,which comprises providing to said mammal an effective amount of asynergistic combination comprising CCI-779 and EKB-569, wherein eitherCCI-779, EKB-569, or both are provided in subtherapeutically effectiveamounts.
 24. The method according to claim 23 in which CCI-779 isprovided in a subtherapeutically effective amount.
 25. The methodaccording to claim 23 in which EKB-569 is provided in asubtherapeutically effective amount.
 26. The method according to claim23 in which both CCI-779 and EKB-569 are provided in subtherapeuticallyeffective amounts.
 27. An antineoplastic combination which comprises anantineoplastic effective amount of a synergistic combination of CCI-779and EKB-569.
 28. A process for the preparation of 4-dimethylamino-but- 2-enoic acid [4 -( 3 -chloro- 4 -fluoro-phenylamino)- 3 -cyano- 7-ethoxy-quinolin- 6 -yl]-amide comprising: (a) forming a mixturecontaining ( 4 -[3 -chloro- 4 -fluoro-phenyl]amino- 6 -amino- 3 -cyano-7 -ethoxy-quinoline), 4 -N,N-dimethylaminocrotonoyl chloridehydrochloride, and N-methylpyrrolidinone at a temperature of 0 to 10°C.; (b) holding the mixture until the concentration of the ( 4 -[3-chloro- 4 -fluoro-phenyl]amino- 6 -amino- 3 -cyano- 7-ethoxy-quinoline) is equal to or less than 2 %; (c) adding sodiumbicarbonate to the suspension in step (b) until a solution is obtained;(d) collecting the product of step (c) to yield the compound 4-dimethylamino-but- 2 -enoic acid [4 -( 3 -chloro- 4-fluoro-phenylamino)- 3 -cyano- 7 -ethoxy-quinolin- 6 -yl]-amide. 29.The process according to claim 28 wherein the collecting step comprisesfiltering, washing and drying the product of step (c).
 30. The processaccording to claim 28, further comprising the step of preparing the4-N,N-dimethylaminocrotonoyl chloride hydrochloride, said step furthercomprising: (i) adding oxalyl chloride to a combination of the compound

(ii) stirring the combination in step (i) at 20 to 25° C.; (iii) coolingthe combination of step (ii).
 31. The process according to claim 30,wherein the combination in (i) is at 0° C.
 32. The process according toclaim 30, wherein the cooling is to 0° C.
 33. The process according toclaim 30, wherein the product of (iii) is washed with tetrahydrofuran(THF) and dried to yield 4 -N,N-dimethylaminocrotonoyl chloridehydrochloride.
 34. The process according to claim 28, wherein the4-N,N-dimethylaminocrotonoyl chloride hydrochloride is prepared in CH ₃CN.
 35. The process according to claim 28, wherein the4-N,N-dimethylaminocrotonoyl chloride hydrochloride is prepared intetrahydrofuran with dimethylformamide.